The ability to ‘tether’ or ‘immobilise’ small molecules at a desired location is important for numerous applications. For example, in biological systems, the immobilisation of proteins at a solid substrate may be used to isolate and/or detect specific proteins within a complex biological sample. Moreover, the tethering of a protein to a substrate is often used to achieve separation or purification of proteins from a mixture of biological molecules, for example a cell lysate.
Several approaches exist for the immobilisation, detection and purification of biological molecules, including proteins. For example, antibodies may be exploited to purify their respective antigens by immunoprecipitation. Proteins may also be purified using alternative affinity purification techniques, wherein an “affinity ligand” capable of binding to the protein of interest is typically used to isolate the protein.
For the purposes of affinity purification, the protein of interest is often “tagged” with a molecule to which an affinity ligand specifically binds. Several molecular tagging systems have been developed and used to generate fusion proteins incorporating tags including the following: myc tag; Flag-peptide tag; His Tag; Strep-Tag; GST-Tag; MBP-Tag; SNAP-Tag; Halo-Tag; Tap-Tag; INPACT-CN. The cognate affinity ligands for each of these tags are known and can be used, for example, in the context of affinity chromatography approaches, for the isolation of tagged proteins of interest.
Despite the availability of several commercial molecular tagging systems, there are disadvantages associated with many of the existing tags. In particular, the size of the tag can create problems during production of the recombinant fusion protein, such as the formation of inclusion bodies, difficulty in solubilisation, lack of stability and/or incorrect folding of the fusion protein and non-specific purification of bacterial proteins. Moreover, it has been reported that metal containing resins that bind His-Tags promote non-specific oxidation on amino acid side chains of the protein during purification. This oxidation often affects protein functionality.
An additional challenge in the field of molecular or affinity tags used for the purposes of protein purification is to balance the strength and specificity of binding needed to achieve efficient purification with a sufficiently low-affinity interaction that can be dissociated so as to elute the purified protein. If the strength of binding between the tag of the fusion protein and its cognate affinity ligand is too high, a harsh elution protocol may be required to release the protein, and this may significantly impair the function of the purified protein.